Measurement of human latent Transforming Growth Factor-β1 using a latency associated protein-reactive ELISA

Abstract

Human Transforming Growth Factor (TGF)-β1, one of three TGF-β isoforms, is a pleotropic cytokine critical for many physiological and immunological processes. TGF-β1 is secreted in a latent form, linked to Latency Associated Protein (LAP). Analysis of Latent TGF-β1 by TGF-β1 ELISA requires dissociation of TGF-β1 from LAP, e.g. by acidification of samples. The ELISA then measures total TGF-β1, equivalent to dissociated Latent TGF-β1 plus any free TGF-β1 present prior to acidification. Evolutionary conservation of TGF-β1 across mammals also renders TGF-β1 ELISAs reactive with TGF-β1 in bovine serum often used in human cell cultures. To enable a direct analysis of Latent TGF-β1, monoclonal antibodies were made against LAP from human Latent TGF-β1 and used to develop a LAP ELISA detecting Latent TGF-β1. The ELISA did not react with LAP from human Latent TGF-β2 or 3, respectively, nor with Latent TGF-β in bovine serum. EDTA-containing plasma from healthy subjects (n=20) was analyzed by conventional TGF-β1 ELISA and LAP ELISA. By TGF-β1 ELISA, total TGF-β1 were detected in all samples (median 133 pM, range 34-348 pM); low levels of free TGF-β1 found in 8/20 non-acidified samples showed that >98.5% of the total TGF-β1 derived from Latent TGF-β1. Latent TGF-β1 found in non-acidified samples by LAP ELISA (median 154 pM, range 48-403 pM) was comparable in molar levels to, and correlated with, total TGF-β1 (r(s) 0.96, p<0.0001). A similar agreement between the total TGF-β1 and the LAP ELISA was found with citrate- and heparin-containing plasma. The LAP ELISA facilitates analysis of Latent TGF-β1 without sample acidification and is not compromised by the presence of bovine serum in human cell supernatants.